阅读说明：本技术 一种检测石斑鱼神经坏死病毒的胶体金试纸条及其制备和检测方法 (Colloidal gold test strip for detecting nervous necrosis virus of grouper and preparation and detection methods thereof ) 是由 秦启伟 刘嘉昕 王劭雯 曾令文 俞也频 李趁 魏世娜 于 2019-09-17 设计创作，主要内容包括：本发明涉及病毒检测技术领域,公开了一种检测石斑鱼神经坏死病毒的胶体金试纸条及其制备方法,所述胶体金试纸条包括底板以及依次搭接固定于底板上的样品垫、结合垫、硝酸纤维素膜以及吸收垫,所述结合垫上包被有金标探针,所述硝酸纤维素膜上设置有检测线和质控线,所述检测线固定有捕获探针,所述质控线固定有质控探针。本发明同时公开了一种采用上述胶体金试纸条检测石斑鱼神经坏死病毒的方法。本发明的胶体金试纸条基于核酸适配体结合侧流生物传感器以及胶体金显色的原理制成,具有较高的灵敏度和特异性,组装简单,便于携带,检测方法操作简便,可以对石斑鱼神经坏死病毒进行高效、准确、快速的现场实时检测。(the invention relates to the technical field of virus detection, and discloses a colloidal gold test strip for detecting a grouper nervous necrosis virus and a preparation method thereof. The invention also discloses a method for detecting the nervous necrosis virus of the grouper by adopting the colloidal gold test strip. The colloidal gold test strip is prepared based on the principles of aptamer combination with a lateral flow biosensor and colloidal gold color development, has high sensitivity and specificity, is simple to assemble and carry, is simple and convenient to operate, and can be used for efficiently, accurately and quickly detecting the nervous necrosis virus of the grouper on site in real time.)

1. A colloidal gold test strip for detecting the nervous necrosis virus of grouper comprises a base plate, a sample pad, a combination pad, a nitrocellulose membrane and an absorption pad which are sequentially lapped and fixed on the base plate,

the combination pad is coated with a gold-labeled probe, the gold-labeled probe is a nucleic acid aptamer modified by 5' end sulfydryl, and the nucleotide sequence of the gold-labeled probe is as follows: 5' -SH-AGCTACTGCTTTGGGGGT;

the nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is fixed with a capture probe, and the quality control line is fixed with a quality control probe:

the nucleotide sequence of the capture probe is as follows: TTCTTTTATTAGTTGATTT, respectively;

the nucleotide sequence of the quality control probe is as follows: ACCCCCAAAGCAGTAGCT are provided.

2. the method for preparing the colloidal gold test strip for detecting the nervous necrosis virus of the grouper as claimed in claim 1, wherein the method comprises the following steps:

S1, preparing a sample pad;

S2, preparing a bonding pad, and coating a gold-labeled probe on the bonding pad;

s3, processing of the nitrocellulose membrane: marking the capture probe and the quality control probe on the nitrocellulose membrane by a gold spraying instrument according to the sample spraying amount of 0.5-0.8 mu L/cm to obtain a detection line and a quality control line, wherein the distance between the detection line and the quality control line is 5-6 mm;

S4, assembling the colloidal gold test strip: and fixing the sample pad, the combination pad, the nitrocellulose membrane and the absorption pad on the bottom plate in sequence, overlapping each adjacent part for 2-3 mm, cutting the whole body into 4mm wide, and storing in a dark place.

3. the method for preparing a colloidal gold test strip for detecting the nervous necrosis virus of grouper according to claim 2, wherein the S1 is specifically: soaking glass fiber in solution containing 0.5% Triton X-100, 2% sucrose, 1% Bovine Serum Albumin (BSA), 50mM boric acid, pH 8.0, and drying at room temperature for 12 hr.

4. the method for preparing the colloidal gold test strip for detecting the nervous necrosis virus of grouper according to claim 2, wherein the S2 specifically comprises the following steps:

a. Synthesis of colloidal gold: 200ml of 0.01% HAuCl were taken₄Stirring and heating the solution to boiling, adding 8ml of 1% trisodium citrate, continuing to boil for 5min when the solution turns red, stopping heating, stirring and cooling to room temperature to obtain colloidal gold solution;

b. Preparation of colloidal gold-aptamer complex: concentrating the colloidal gold solution obtained in the step I by 10 times, adding 400 mu l of concentrated solution into a gold-labeled probe, and shaking at 4 ℃ for 12 hours; then adding BSA with the concentration of 10% to react for 4 hours to obtain a colloidal gold-aptamer compound;

Adding 1% of sodium dodecyl sulfate into the compound until the final concentration is 0.01%, then adding 1.5mol/L of NaCl solution to enable the final concentration of salt ions to be 150mM, and placing the obtained compound solution at 4 ℃ for reaction for 12 hours;

c. Centrifuging the composite solution obtained in the step b at the rotating speed of 12000rpm for 10-20 min, washing the composite solution with a buffer solution for three times, discarding the supernatant, suspending the obtained particles in 150 mu l of the buffer solution to obtain a colloidal gold-aptamer binding solution, and storing the colloidal gold-aptamer binding solution at 4 ℃;

d. and d, dropwise adding 2-3 mu l of the colloidal gold-aptamer combined solution obtained in the step c on the combined pad, and air-drying at room temperature for 5min to obtain the combined pad coated with the gold-labeled probe.

5. The colloidal gold test strip for detecting the nervous necrosis virus of grouper as claimed in claim 4, wherein the buffer solution in step c is 20mM Na₃PO₄5% BSA, 0.25% Tween and 10% sucrose.

6. the method for detecting the colloidal gold test strip for detecting the nervous necrosis virus of the grouper as claimed in any one of claims 1 to 5, wherein the method comprises the following steps:

Virus incubation: adding a capture Aptamer (Aptamer1) and an amplification Aptamer (Aptamer2) into 100 mu l of phosphate buffer solution to enable the concentrations of the capture Aptamer and the amplification Aptamer to be 200nM respectively, and adding a cell lysate infected by the grouper nervous necrosis virus (RGNNV) for incubation for 10-15 min to obtain a mixture containing the RGNNV;

② strand displacement reaction: adding 2 mu l of magnetic beads modified by streptavidin into the mixture, oscillating for 10-15 min to obtain an Aptamer1-RGNNV-Aptamer 2-magnetic bead compound, collecting the compound by using a magnetic separator separation frame, washing the compound three times by using PBST, transferring the compound into a centrifuge tube for chain displacement reaction to obtain a chain displacement reaction product;

sampling and detecting: dropwise adding the chain displacement reaction product obtained in the step two on a sample pad of the colloidal gold test strip, reacting for 5min, and observing the color development result of the colloidal gold test strip;

fourthly, interpretation of results:

positive reaction: the detection line and the quality control line of the colloidal gold test strip are colored;

negative reaction: the detection line of the colloidal gold test strip is not colored, and the quality control line is colored.

7. The method for detecting the colloidal gold test strip of the nervous necrosis virus of grouper as claimed in claim 6, wherein the temperature of the strand displacement reaction in the second step is 37 ℃ and the time is 30 min.

8. the method for detecting the colloidal gold test strip for detecting the nervous necrosis virus of grouper as claimed in claim 7, wherein the strand displacement reaction system in the step (II) comprises, by volume: SDA primer 2. mu.l, deoxynucleoside triphosphate (dNTPs) 2. mu.l, polymerase (Klenow-exo) 0.6. mu.l, nicking endonuclease (Nt. BbvCI) 0.4. mu.l, SDA-buffer-22. mu.l, 10% BSA 1. mu.l, and ultrapure water 12. mu.l.

9. The method for detecting the colloidal gold test strip for detecting the nervous necrosis virus of the grouper as claimed in claim 8, wherein the nucleotide sequence of the SDA primer is as follows: GAGACTTCATCTGCGTCCTTCG are provided.

10. The method for detecting the colloidal gold test strip for detecting the nervous necrosis virus of the grouper as claimed in any one of claims 6 to 9, wherein the sequence of the capture Aptamer (Aptamer1) is as follows: TTCTTTTATTAGTTGATTTTTTTGATTTTGGCAGCTACTGCTTTGGGGGT-Bio;

The sequence of the amplification Aptamer (Aptamer2) is as follows: TTCTTTTATTAGTTGATTTTTTTGATTTTGGCAGCTACTGCTTTGGGGGTGCTGAGGCGAAGGACGCAGATGAAGTCTC are provided.

Technical Field

the invention relates to the technical field of virus detection, in particular to a colloidal gold test strip for detecting nervous necrosis virus of grouper and a preparation method and a detection method thereof.

Background

the nervous necrosis virus belongs to beta-nodavirus of nodaviridae, constitutes a great threat to aquaculture industry, mainly aims at the infection of the central nervous system of lower vertebrates, and can observe obvious vacuole phenomenon in retina and brain of fish infected by the nervous necrosis virus. The nervous necrosis virus (RGNNV) of grouper, the nervous necrosis virus (SJNNV) of scad, the nervous necrosis virus (BFNNV) of spotted halibut and the nervous necrosis virus (TPNNV) of Takifugu rubripes belong to four genotypes of the nervous necrosis virus, and the RGNNV is the most serious.

the grouper is a fish having great economic value, the death rate of larval and juvenile fish after being infected by the nervous necrosis virus can reach 100%, and more seriously, the nervous necrosis virus has the migration ability to transfer from the infected grouper to the offspring. RGNNV causes destructive damage to groupers and causes huge economic loss to aquaculture. Therefore, it is necessary to establish an effective diagnostic method for early detection and prevention of such viral diseases.

among the current methods for detecting a nervous necrosis virus, cell culture is the most basic culture method, but it is time-consuming and temperature-dependent; the conventional polymerase chain reaction as a standard method has been developed into various forms such as reverse transcription polymerase chain reaction (RT-PCR), real-time quantitative RT-PCR (qRT-PCR), loop-mediated isothermal amplification (LAMP), etc., but although the polymerase chain reaction has been widely used, there are some disadvantages such as the need for complicated procedures and dedicated equipment, which cannot be used in the field. Other detection methods, such as electron microscopy of infected tissues, enzyme-linked immunosorbent assay (ELISA), and immunofluorescence antibody assay (IFAT), are difficult to meet with real-time detection requirements due to the need for professional operation and expensive instrumentation.

In summary, it is necessary to develop a colloidal gold test strip for detecting the nervous necrosis virus of grouper and a preparation and detection method thereof.

Disclosure of Invention

the colloidal gold test strip for detecting the nervous necrosis virus of the grouper and the preparation and detection methods thereof solve the problems that the existing nervous necrosis virus detection method is time-consuming and labor-consuming, high in detection condition requirement, complex in detection procedure and incapable of performing real-time detection on site. The colloidal gold test strip and the detection method are based on the principle that the aptamer is combined with a lateral flow biosensor and the colloidal gold is developed, have high sensitivity and specificity, are simple to prepare and assemble, are convenient to carry, are simple in detection method and convenient to operate, and can be used for efficiently, accurately and quickly detecting the nervous necrosis virus of the grouper.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

the utility model provides a detect colloidal gold test paper strip of grouper nervous necrosis virus, the colloidal gold test paper strip includes the bottom plate and sample pad, combination pad, nitrocellulose membrane and the absorption pad that overlap joint was fixed in on the bottom plate in proper order, the parcel has the gold mark probe on the combination pad, the gold mark probe is the nucleic acid aptamer of 5' end mercapto modification, and its nucleotide sequence is: 5' -SH-AGCTACTGCT TTGGGGGT (SEQ ID NO: 1);

the nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is fixed with a capture probe, and the quality control line is fixed with a quality control probe:

The nucleotide sequence of the capture probe is as follows: TTCTTTTATTAGTTGATTT (SEQ ID NO: 2);

The nucleotide sequence of the quality control probe is as follows: ACCCCCAAAGCAGTAGCT (SEQ ID NO: 3).

the invention also discloses a preparation method of the colloidal gold test strip for detecting the nervous necrosis virus of the grouper, which comprises the following steps:

S1, preparing a sample pad;

s2, preparing a bonding pad, and coating a gold-labeled probe on the bonding pad;

S3, processing of the nitrocellulose membrane: scribing a capture probe and a quality control probe on a nitrocellulose membrane by adopting a three-dimensional scribing and gold spraying instrument according to a sample spraying amount of 0.5-0.8 mu L/cm to obtain a detection line and a quality control line, wherein the distance between the detection line and the quality control line is 5-6 mm;

s4, assembling the colloidal gold test strip: and fixing the sample pad, the combination pad, the nitrocellulose membrane and the absorption pad on the bottom plate in sequence, overlapping each adjacent part for 2-3 mm, cutting the whole body into pieces with the width of 4mm, and storing the pieces for later use in a dark closed environment.

Further, the step S1 is specifically: soaking glass fiber in solution containing 0.5% polyethylene glycol octyl phenyl ether (Triton X-100), 2% sucrose, 1% BSA, and 50mM boric acid at pH 8.0, and drying at room temperature for 12 hr.

Further, the step S2 specifically includes the following steps:

a. synthesis of colloidal gold (AuNP): placing 200ml HAuCl4 solution with concentration of 0.01% into 500ml round bottom flask, stirring gently and heating to boil, rapidly adding 8ml trisodium citrate with concentration of 1%, wherein the solution color is changed into dark blue, then into wine red, boiling for 5min, and stirring gently; stopping heating, moving to a magnetic stirrer after 15min, stirring and cooling to room temperature to obtain an AuNP solution;

b. preparation of colloidal gold-aptamer (AuNP-DNA) complexes: concentrating the AuNP solution obtained in the step (i) by 10 times, adding a gold-labeled probe into 400 mul of concentrated solution, slightly shaking for 12 hours at 4 ℃, then adding Bovine Serum Albumin (BSA) with the concentration of 10%, and reacting for 4 hours to obtain a colloidal gold-aptamer (AuNP-DNA) compound; adding 1% sodium dodecyl sulfate into the compound until the final concentration of the sodium dodecyl sulfate in the system is 0.01%, then adding 1.5mol/L NaCl solution to enable the final concentration of salt ions in the system to be 150mM, and placing the obtained compound solution at 4 ℃ for reaction for 12 hours;

c. Centrifuging the composite solution obtained in the step b at the rotating speed of 12000rpm for 10-20 min, washing the composite solution with a buffer solution for three times, removing redundant unbound DNA, discarding the supernatant, resuspending the obtained red particles in 150 mu l of the buffer solution to obtain a colloidal gold-aptamer (AuNP-DNA) binding solution, and storing the colloidal gold-aptamer (AuNP-DNA) binding solution at 4 ℃;

d. and d, dripping the colloidal gold-aptamer binding solution obtained in the step c on the binding pad in an amount of 2-3 mu l per strip, and air-drying at room temperature for 5min to obtain the binding pad coated with the gold-labeled probe.

further, the buffer solution in step c contains 20mM Na₃PO₄5% BSA, 0.25% Tween and 10% sucrose.

the invention also discloses a detection method for detecting the nervous necrosis virus of the grouper by adopting the colloidal gold test strip, which comprises the following steps:

Virus incubation: adding a capture Aptamer (Aptamer1) and an amplification Aptamer (Aptamer2) into 100 mu l of Phosphate Buffer Solution (PBS) to enable the concentrations of the capture Aptamer (Aptamer1) and the amplification Aptamer (Aptamer2) to reach 200nM respectively, and adding a grouper nervous necrosis virus (RGNNV) infected cell lysate for incubation for 10-15 min to obtain a mixture containing the grouper nervous necrosis virus (RGNNV);

② strand displacement reaction: adding 2 mu l of magnetic beads modified by Streptavidin (SA) into the mixture, oscillating for 10-15 min to obtain an Aptamer1-RGNNV-Aptamer 2-magnetic bead compound, collecting the Aptamer1-RGNNV-Aptamer 2-magnetic bead compound by using a magnetic separator separation frame, washing the compound for three times by using a Phosphate Buffer Solution (PBST) containing Tween-20, transferring the compound into a centrifuge tube to perform chain displacement reaction (SDA) at the reaction temperature of 37 ℃ for 30min to obtain an SDA reaction product;

Sampling and detecting: dripping 3-4 SDA reaction products obtained in the second step onto a sample pad of the colloidal gold test strip, reacting for 5min, and observing a color development result of the colloidal gold test strip;

Fourthly, interpretation of results:

Positive reaction: the detection line and the quality control line of the colloidal gold test strip are colored;

negative reaction: the detection line of the colloidal gold test strip is not colored, and the quality control line is colored.

Further, the strand displacement reaction (SDA) reaction system in the second step includes, by volume: SDA primer 2. mu.l, deoxynucleoside triphosphate (dNTPs) 2. mu.l, polymerase (Klenow fragment exo) 0.6. mu.l, nicking endonuclease (Nt. BbvCI) 0.4. mu.l, SDA-buffer-22. mu.l, 10% BSA 1. mu.l, and ultrapure water 12. mu.l.

further, the nucleotide sequence of the SDA primer is as follows: GAGACTTCATCTG CGTCCTTCG (SEQ ID NO: 7).

Further, the capture Aptamer (Aptamer1) and the amplification Aptamer (Aptamer2) are prepared by:

Screening for viral aptamers: adopting a magnetic bead method SELEX technology to screen and obtain the aptamer of the grouper nervous necrosis virus capsid protein (RGNNV-CP for short), wherein the nucleotide sequence of the aptamer is as follows: TTCTTTTATTAGTTGATTTTTTT GATTTTGGCAGCTACTGCTTTGGGGGT (SEQ ID NO: 4);

nucleic acid aptamer modification: carrying out 3' end biotin modification on the Aptamer in the step I to obtain a capture Aptamer (Aptamer1), wherein the sequence of the capture Aptamer is as follows: TTCTTTTATTAGTTGATTTTTTTGATTTTGGCAGCTACTGCTTTGGGGGT-Bio (SEQ ID NO: 5);

Modifying the recognition site of a nicking endonuclease (Nt.BbvCI) and the primer binding site of the Aptamer of the step I to obtain an amplified Aptamer (Aptamer2), wherein the sequence of the amplified Aptamer is as follows: TTCTTTTATTAGTTGATTTTTTTGATTTTGGCAGCTACTGCTTTGGGGGTGCTGAGGCGAAGGACGCAGATGAAGTCTC (SEQ ID NO: 6);

in the amplification Aptamer (Aptamer2) sequence, "GCTGAGG" is the recognition site for nicking endonuclease (nt.bbvci) and "CGAAGGACGCAGATGAAGTCTC" is the primer binding sequence.

The optimized screening and detection principle of the colloidal gold test strip and the detection method thereof is as follows:

culturing the grouper brain cells infected by RGNNV in a culture medium, preparing a grouper nervous necrosis virus capsid protein (RGNNV-CP for short), then optimally screening the RGNNV-CP by a magnetic bead method SELEX technology, constructing a single-stranded oligonucleotide library containing different sequences by chemical synthesis, incubating magnetic beads with the RGNNV-CP fixed on the surface together with the library so as to ensure that a target is fully combined with corresponding single-stranded nucleic acid, then obtaining a nucleic acid single strand which is specifically combined with the target on the surface of the magnetic beads by a magnetic separation procedure, then eluting the combined nucleic acid by an elution procedure, amplifying by using the eluted nucleic acid as a template and using the amplified nucleic acid as the next round of screening, eliminating the nucleic acid which is not combined with the target and has smaller affinity by repeatedly combining, separating, eluting and amplifying operation steps, and gradually enriching the nucleic acid aptamer with high affinity, finally, the aptamer with specific binding force with RGNNV-CP is screened out.

performing biotin modification on the Aptamer of the screened RGNNV-CP to obtain a capture Aptamer (Aptamer1), wherein the capture Aptamer (Aptamer1) has biotin modification and can be captured by Streptavidin (SA) modified magnetic beads so as to be used for enrichment; the amplified Aptamer (Aptamer2) can be obtained by modifying the Aptamer of RGNNV-CP with Nt. BbvCI enzyme recognition site and primer binding site, and the amplified Aptamer (Aptamer2) can be used for amplification and can be used as a template for strand displacement reaction (SDA).

the Aptamer1 and Aptamer2 were incubated with RGNNV in Phosphate Buffered Saline (PBS), SA-modified magnetic beads were added to the resulting mixture, then Aptamer1-RGNNV-Aptamer 2-magnetic bead complexes were collected with a magnetic separator stand and washed with Phosphate Buffered Saline (PBST) containing Tween-20, and then the Aptamer1-RGNNV-Aptamer 2-magnetic bead complexes were transferred to a centrifuge tube with ultrapure water for SDA reaction.

in the SDA reaction process, the Aptamer2 is combined with the SDA primer, the formed complementary strand is cut and separated at the recognition site of the Nt.BbvCI enzyme, the amplification reaction is continuously carried out from the cutting site under the action of polymerase, the amplification reaction is continuously cut and separated, the cycle is repeated, after the SDA reaction, a large number of amplification products are generated, then the amplification products are dripped on a sample pad of a colloidal gold test strip, and the amplification products flow through a detection line and a quality control line through the chromatography action and carry out the complementary reaction with a capture probe and a quality control probe.

In summary, in the presence of RGNNV-CP, the Aptamer1-RGNNV-Aptamer 2-magnetic bead complex first undergoes SDA reaction to obtain ssDNA, and the gold-labeled probe in the AuNP-DNA complex and the SDA product (ssDNA) undergo complementary reaction on the binding pad of the colloidal gold test strip, so that the AuNP-DNA complex captures ssDNA to form ssDNA-AuNP-DNA complex.

because the ssDNA is also complementary to the capture probe on the detection line, the formed ssDNA-AuNP-DNA complex can be gathered on the detection line to form a visible red band; excessive AuNP-DNA compound continues to migrate on the nitrocellulose membrane, and another red band is formed on the quality control line because the gold-labeled probe in the AuNP-DNA compound and the quality control probe on the quality control line can carry out complementary reaction.

Compared with the prior art, the technical scheme of the invention has the beneficial effects that:

the colloidal gold test strip is prepared based on the principle that the aptamer is combined with a lateral flow biosensor and colloidal gold develops color, because the aptamer can be folded into a three-dimensional structure and is further combined with a target substance through space configuration complementation or intermolecular acting force, various target spots can be combined, including organic molecules, proteins, cells and metal ions, the colloidal gold test strip has high specificity and the same characteristic as an antibody, the identification mode of the aptamer and the target substance is similar to that of the antibody, but compared with the traditional protein antibody, the aptamer is essentially oligonucleotide chains and is far smaller than the antibody, so that the colloidal gold test strip can identify and distinguish slight difference on the structure of the target molecule, is more stable and cheaper, and is easy to synthesize through modification. Therefore, the colloidal gold test strip has the advantages of high sensitivity and specificity, stable performance, easy preservation and standby, simple preparation and assembly, low cost, convenient carrying, on-site real-time detection and convenient use.

The method for detecting the grouper nervous necrosis virus (RGNNV) by using the colloidal gold test strip has the advantages of simple steps, convenient operation and short detection time, is suitable for field real-time detection, and can efficiently, accurately and quickly detect the grouper nervous necrosis virus, so that the early morbidity is known in time, destructive damage is prevented, and the grouper culture risk is reduced, thereby having good application prospect.

Drawings

FIG. 1 is a schematic structural diagram of a colloidal gold test strip of the present invention;

FIG. 2 is a diagram showing the test result of the colloidal gold test strip of the present invention;

FIG. 3 is a graph showing the results of a comparative experiment of the colloidal gold test strip of comparative example 1 according to the present invention;

FIG. 4 is a graph showing the results of a comparative experiment of the colloidal gold test strip of comparative example 2 of the present invention;

FIG. 5 is a graph showing the results of a comparative experiment of the colloidal gold test strip of comparative example 3 of the present invention.

in the figure: 1-bottom plate, 2-sample pad, 3-combination pad, 4-nitrocellulose membrane, 41-detection line, 42-quality control line, and 5-absorption pad.

Detailed Description

The present invention is further illustrated by the following specific examples, which are presently preferred embodiments of the invention, but are not intended to limit the scope of the invention, as claimed.

Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the materials, reagents and the like used are commercially available. The nucleotide sequences in the present invention were optimally designed by the inventors through a large number of experiments and then synthesized by Invitrogen Biotechnology co.ltd (shanghai, china).

1. main reagents and materials:

HPLC purified oligonucleotides were synthesized by Invitrogen Biotechnology co.ltd (shanghai, china);

HAuCl₄purchased from sigmaldrich (germany);

bovine Serum Album (BSA) was purchased from Sigma Aldrich (Germany);

Polymerase (Klenow-exo) was purchased from new england biological laboratory (usa);

Nicking endonuclease (nt. bbvci) was purchased from new england biological laboratory (usa);

SDA-buffer-2 was purchased from New England Biolabs (USA);

Deoxynucleoside triphosphates (dNTPs) were purchased from Takara (beijing, china);

Streptavidin(SA) Modified magnetic beads (DynaBeads)^TMMyone^TMStreptavidin C1) was purchased from Invitrogen (usa);

nitrocellulose membranes were purchased from Millipore, usa;

glass fibers, PVC sole plates and absorbent pads were purchased from oswegian biotechnology limited, guangzhou.

2. example 1:

as shown in fig. 1, a colloidal gold test strip for detecting nervous necrosis virus of grouper, the colloidal gold test strip comprises a base plate 1, and a sample pad 2, a binding pad 3, a nitrocellulose membrane 4 and an absorption pad 5 which are sequentially fixed on the base plate 1 in a lap joint manner, wherein a gold-labeled probe is coated on the binding pad 3, the gold-labeled probe is a nucleic acid aptamer modified by 5' end sulfhydryl, and the nucleic acid aptamer has the nucleotide sequence: 5' -SH-AGCTACTGCTTTGGGGGT (SEQ ID NO: 1);

The nitrocellulose membrane 4 is provided with a detection line 41 and a quality control line 42, the detection line 41 is fixed with a capture probe, and the quality control line 42 is fixed with a quality control probe:

the nucleotide sequence of the capture probe is as follows: TTCTTTTATTAGTTGATTT (SEQ ID NO: 2);

The nucleotide sequence of the quality control probe is as follows: ACCCCCAAAGCAGTAGCT (SEQ ID NO: 3).

The preparation method of the colloidal gold test strip for detecting the nervous necrosis virus of the grouper comprises the following steps:

s1, preparing a sample pad 2: soaking glass fiber in solution containing 0.5% polyethylene glycol octyl phenyl ether (Triton X-100), 2% sucrose, 1% BSA, 50mM boric acid, pH 8.0, and drying at room temperature for 12 hr to obtain the final product;

s2, preparing a bonding pad 3, and coating a gold-labeled probe on the bonding pad;

s3, treating the nitrocellulose membrane 4: scribing a capture probe and a quality control probe on the nitrocellulose membrane 4 by adopting a three-dimensional scribing and gold spraying instrument according to the spraying amount of 0.6 mu L/cm to obtain a detection line 41 and a quality control line 42, wherein the distance between the detection line 41 and the quality control line 42 is 6 mm;

s4, assembling the colloidal gold test strip: the sample pad 2, the combination pad 3, the nitrocellulose membrane 4 and the absorption pad 5 are sequentially assembled on the base plate 1, the adjacent components are overlapped by 2mm, and then the whole is cut into 4mm wide by a paper cutter and stored for standby under a dark and closed environment.

wherein, the step S2 specifically includes the following steps:

a. Synthesis of colloidal gold (AuNP): placing 200ml HAuCl4 solution with concentration of 0.01% into 500ml round bottom flask, stirring gently and heating to boil, rapidly adding 8ml trisodium citrate with concentration of 1%, wherein the solution color is changed into dark blue, then into wine red, boiling for 5min, and stirring gently; stopping heating, moving to a magnetic stirrer after 15min, stirring and cooling to room temperature to obtain an AuNP solution;

b. Preparation of colloidal gold-aptamer (AuNP-DNA) complexes: concentrating the AuNP solution obtained in the step (i) by 10 times, adding a gold-labeled probe into 400 mul of concentrated solution, slightly shaking for 12 hours at 4 ℃, then adding Bovine Serum Albumin (BSA) with the concentration of 10%, and reacting for 4 hours to obtain a colloidal gold-aptamer (AuNP-DNA) compound; adding 1% sodium dodecyl sulfate into the compound until the final concentration of the sodium dodecyl sulfate in the system is 0.01%, adding 1.5mol/L NaCl solution to enable the final concentration of salt ions in the system to be 150mM, and placing the obtained compound solution at 4 ℃ for reaction for 12 hours;

c. centrifuging the composite solution obtained in the step b at the rotating speed of 12000rpm for 20min, washing the composite solution with a buffer solution for three times, removing redundant unbound DNA, discarding the supernatant, and resuspending the obtained red particles in 150 mu l of the buffer solution to obtain a colloidal gold-aptamer (AuNP-DNA) binding solution, and storing the colloidal gold-aptamer (AuNP-DNA) binding solution at 4 ℃; the buffer solution contains 20mM Na₃PO₄a solution of 5% BSA, 0.25% tween and 10% sucrose;

d. And d, dripping the colloidal gold-aptamer binding solution obtained in the step c on the binding pad in an amount of 2.5 mu l per strip, and air-drying at room temperature for 5min to obtain the binding pad coated with the gold-labeled probe.

As shown in fig. 3-5, for convenience of carrying and use, a specially-made packing box can be designed for the colloidal gold test strip, the colloidal gold test strip is embedded in the packing box, a sample adding hole is arranged on the lower section of the packing box, the sample adding hole is directed at a sample pad, a transparent window is arranged on the upper end of the packing box, a detection line and a quality control line of the colloidal gold test strip can be seen, and a color development result can be observed conveniently.

3. example 2:

The method for detecting the nervous necrosis virus of the grouper by adopting the colloidal gold test strip comprises the following steps:

Virus incubation: adding capture Aptamer (Aptamer1) and amplification Aptamer (Aptamer2) into 100 μ l of Phosphate Buffer Solution (PBS) to make the concentrations of the capture Aptamer (Aptamer1) and the amplification Aptamer (Aptamer2) 200nM respectively, and adding cell lysate infected by the grouper nervous necrosis virus (RGNNV) for incubation for 15min to obtain a mixture containing the grouper nervous necrosis virus (RGNNV);

② strand displacement reaction: adding 2 mu l of magnetic beads modified by Streptavidin (SA) into the mixture, oscillating for 15min to obtain an Aptamer1-RGNNV-Aptamer 2-magnetic bead complex, collecting the Aptamer1-RGNNV-Aptamer 2-magnetic bead complex by using a magnetic separator stand, washing the complex three times by using Phosphate Buffer Solution (PBST) containing Tween-20, transferring the complex into a centrifuge tube (EP tube) to perform chain displacement reaction (SDA), wherein the reaction temperature is 37 ℃ and the time is 30min, and obtaining a chain displacement reaction (SDA) product;

sampling and detecting: dripping 3-4 of the reaction product obtained in the second step onto a sample pad of the colloidal gold test strip, reacting for 5min, and observing a color development result of the colloidal gold test strip;

Fourthly, interpretation of results:

as shown in fig. 2, positive reaction: the detection line and the quality control line of the colloidal gold test strip are colored; negative reaction: the detection line of the colloidal gold test strip is not colored, and the quality control line is colored.

the strand displacement reaction (SDA) reaction system in the step II comprises the following components in volume: SDA primer 2. mu.l, deoxynucleoside triphosphate (dNTPs) 2. mu.l, nicking endonuclease (Nt. BbvCI) 0.4. mu.l, polymerase (Klenow fragmentexo) 0.6. mu.l, SDA-buffer-22. mu.l, 10% BSA 1. mu.l, and ultrapure water 12. mu.l.

Wherein the nucleotide sequence of the SDA primer is as follows: GAGACTTCATCTGCGTCCTTCG (SEQ ID NO: 7).

the capture Aptamer (Aptamer1) and the amplification Aptamer (Aptamer2) are prepared by the following steps:

screening for aptamers: adopting a magnetic bead method SELEX technology to screen and obtain the aptamer of the grouper nervous necrosis virus capsid protein (RGNNV-CP for short), wherein the nucleotide sequence of the aptamer is as follows: TTCTTTTATTAGTTGATTTTTTTGATTTTGGCAGCTACTGCTTTGGGGGT (SEQ ID NO: 4);

nucleic acid aptamer modification: carrying out 3' end biotin modification on the Aptamer in the step I to obtain a capture Aptamer (Aptamer1), wherein the sequence of the capture Aptamer is as follows: TTCTTTTATTAGTTGATTTTTTTGATTTTGGCAGCTACTGCTTTGGGGGT-Bio (SEQ ID NO: 5);

Modifying the Aptamer of the step I with an Nt.BbvCI enzyme recognition site and a primer binding site to obtain an amplified Aptamer (Aptamer2), wherein the sequence of the amplified Aptamer is as follows: TTCTTTTATTAGTTGATTTTTTTGATTTTGGCAGCTACTGCTTTGGGGGTGCTGAGGCGAAGGACGCAGATGAAGTCTC (SEQ ID NO: 6);

in the amplified Aptamer (Aptamer2) sequence, the "GCTGAGG" fragment is the recognition site for the nt. bbvcci enzyme, and the "CGAAGGACGCAGATGAAGTCTC" fragment is the primer binding sequence.

4. Comparative example 1

In order to analyze the feasibility and the effectiveness of the colloidal gold test strip and the detection method, a comparison experiment 1 is specially set, an experiment group and comparison groups 1-3 are specifically set in the comparison experiment 1, and the specific conditions of each group are as follows:

Experimental groups: RGNNV-infected cell lysates were incubated simultaneously with Aptamer1 and Aptamer 2;

Control group 1 (negative control group): phosphate Buffered Saline (PBS) was incubated with Aptamer1 and Aptamer 2;

control group 2: RGNNV-infected cell lysates were incubated with Aptamer1 only;

Control group 3: RGNNV-infected cell lysates were incubated with Aptamer2 only.

after the incubation, the strand displacement reaction (SDA) was performed on each group, and the conditions of the incubation method and the strand displacement reaction (SDA) were the same as those in example 2, and then the reaction products of each group were dropped onto the sample pad of the colloidal gold strip for detection.

as shown in fig. 3, the colloidal gold test strip detection lines (T-line) and the quality control lines (C-line) of the experimental group both show color bands, and the colloidal gold test strip detection lines (T-line) of the control groups 1 to 3 do not show color bands.

Therefore, only in the presence of RGNNV, the Aptamer1-RGNNV-Aptamer 2-magnetic bead complex can firstly carry out SDA reaction to obtain a reaction product ssDNA, and then on the binding pad of the colloidal gold test strip, the gold-labeled probe in the AuNP-DNA complex and the SDA product (ssDNA) carry out complementary reaction, so that the AuNP-DNA complex captures the ssDNA to form the ssDNA-AuNP-DNA complex.

Because the ssDNA is also complementary to the capture probe on the detection line, the formed ssDNA-AuNP-DNA complex can be gathered on the detection line to form a visible red band; excessive AuNP-DNA compound continues to migrate on the nitrocellulose membrane, and another red band is formed on the quality control line because the gold-labeled probe in the AuNP-DNA compound and the quality control probe on the quality control line can carry out complementary reaction.

in the absence of RGNNV or only one of Aptamer1 and Aptamer2, the detection line did not develop color because none of the Aptamer1 was enriched by magnetic beads or no Aptamer2 was amplified to yield SDA product.

Therefore, color development in the detection line (T-line) of the colloidal gold test strip was only seen in the presence of both Aptamer1 and Aptamer2 and RGNNV. Experiments show that the colloidal gold test strip and the detection method can effectively detect the nervous necrosis virus (RGNNV) of the grouper.

5. comparative example 2

In order to analyze the specificity of the colloidal gold test strip and the detection method thereof, a comparison experiment 2 is specially set, and an experiment group and a control group are particularly set in the comparison experiment 2, wherein the specific conditions of each group are as follows:

experimental groups: RGNNV-infected cell lysates were incubated simultaneously with Aptamer1 and Aptamer 2;

control group: MBP is incubated with Aptamer1 and Aptamer2, and the MBP is maltose binding protein.

after the incubation, the strand displacement reaction (SDA) was performed on each group, and the conditions for the incubation method and the strand displacement reaction (SDA) were the same as those in example 2, and then the reaction products of each group were dropped onto a colloidal gold strip for detection.

As shown in fig. 4, the test line (T line) and the quality control line (C line) of the colloidal gold test strip of the experimental group both showed color bands, and the test line (T line) of the colloidal gold test strip of the control group did not develop color.

Therefore, the colloidal gold test strip and the detection method have specificity on the garrupa nervous necrosis virus (RGNNV).

6. Comparative example 3

in order to analyze the sensitivity of the colloidal gold test strip and the detection method, a comparison experiment 3 is specially set, and an experiment group and a control group are particularly set in the comparison experiment 3, wherein the specific conditions of each group are as follows:

experimental groups: RGNNV-infected cell lysates were separately dissolved in PBS to give diluted solutions of different RGNNV concentrations, such as: 1 mug/ml, 500ng/ml, 100ng/ml, 50ng/ml, 10ng/ml, 5 ng/ml;

control group (negative control group): RGNNV was absent from PBS, i.e., the concentration of RGNNV was 0 ng/ml.

After the incubation, the strand displacement reaction (SDA) was performed on each group, and the conditions for the incubation method and the strand displacement reaction (SDA) were the same as those in example 2, and then the reaction products of each group were dropped onto a colloidal gold strip for detection.

as shown in FIG. 5, each colloidal gold test strip showed a color development result for detecting RGNNV at different concentrations, and the color band of the detection line (T line) corresponding to the RGNNV concentration of 5ng/ml was light, but was still observed. Therefore, the lowest concentration of the grouper nervous necrosis virus (RGNNV) observed by the detection line (T line) is 5ng/ml, and the detection line (T line) of the control colloidal gold test strip is not developed, so that the colloidal gold test strip and the detection method have high detection sensitivity.

the above-mentioned embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. It will be apparent to those skilled in the art that other variations and modifications may be made in the foregoing description, and it is not necessary or necessary to exhaustively enumerate all embodiments herein. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.